engleski

Killing of decidual dendritic cells by autologous decidual NK cells

Problem: Possible killing and cytotoxic mechanisms employed by decidual CD56+ cells against autologous CD1a+ and CD83+ dendritic cells (DC) were investigated. Design & methods: Decidual mononuclear cells (DMC), obtained from early human pregnancy decidua after enzymatic digestion and gradient centrifugation were cultured overnight at 37oC. Non adherent DMC fraction was used for positive magnetic separation of CD56+ cells and subsequent enrichment of CD83+ cells from « ; CD56 negative» ; fraction. Decidual CD1a+ cells were separated from decidual adherent cells. NKG2D and NKp46 on CD56+ cells and MIC A/B expression on DC were assessed by immunofluorescence. Cytotoxicity of CD56+ cells against CD1a+ or CD83+ cells were measured by 2-hours PKH-26 (red) cell linker kit and analyzed by flow cytometer. In some cytotoxicity assays monoclonal antibodies (mAb) directed toward cytolytic molecules [perforin, Fas ligand (FasL), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), granulysin], NK cell receptors (NKG2D, NKp46, NKp44, NKp30) or NKG2D ligand - MIC A/B were used, respectively. Mann- Whitney -U test was used for statistical evaluation. Results: Decidual CD56+ cells kill significantly less CD1a+ cells in comparison to CD83+ dendritic cells (p 0.01 at all effector target ratio tested). The addition of anti-perforin mAb completely abrogated CD56 mediated short term cytotoxicity. Additional effects of anti-FasL mAb, anti-TRAIL mAb and anti-granulysin mAb were not observed. NKG2D were expressed on positively selected CD56+ cells. DC expressed negligible percentage of MIC A/B. Pre-treatment of cells with anti-NKG2D or anti MIC A/B mAb did not affect killing of DC. Anti-NKp46 mAb completely abrogated NK cell killing of CD1a+ cells, but not of CD83+ cells. NKp46 was less detected (p= 0.02) on the surface of decidual CD56+ cells after 18 hours contact with CD1a+ cells at cell ratio 5:1, but not after the contact with CD83+ cells in comparison to the cells cultured in the medium only. Conclusion: Decidual CD56+ cells are able to kill both DC subsets by perforin mechanism on NKG2D - MIC A/B independent manner. In comparison to CD83+ cells, decidual CD1a+ cells are less susceptible for NK cell mediated killing, although they are able to bind for NKp46 receptor on decidual NK cells providing that way activating signal in NK cells for their own lyses. Acknowledgement: We gratefully acknowledge the financial support provided by the Croatian Ministry of Science (Grants No. 0620402-0376, 0377 and 0379) and the EMBIC project, European FP6, NoE, LSHM-CT-2004-512040.

cytotoxicity; dendritic cells; NK cells

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